| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Mutation detection methods through phenotype alteration on the target genes always have a possibility to bias the results of the study. Moreover, they cannot be applicable to the mutations generated on un-expressed genes. Thus, it has been expected to find alternative methods to detect gene mutations without using phenotype changes, especially in sensitive, simple and easy procedures. Random mutation capture (RMC) method is one of the candidates, which measures mutation randomly generated on genome without any specific target genes.
After digestion of bacterial genomic DNA with five kinds of restriction enzymes at 37 degree C for 16 hours, the DNA was purified with ethanol precipitation. About 400-bp probe DNA containing the target sequence, 5'-TCGA-3', was prepared by PCR using a 5'-biotynirated primer/un-biotinylated primer set with dUTP, dATP, dCTP and dGTP mixture. The probe was amplified with genomic DNA as template and purified with a microspin column. The biotinylated probe was b
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ound to streptavidin immobilized on magnetic beads for three hours at room temperature. The resultant probe was used as labeled magnetic probe. The digested genome fragments were hybridized with the labeled probe at 37 degree C for 16 hours, then double stranded DNA formed on the beads were captured with magnet and recovered. The double stranded DNAs formed with the target sequence and a labeled probe were treated with TaqI enzyme, which digests the target sequence TCGA, at 65 degree C for one hour, then denatured at 95 degree C for one minute and re-annealed at 50 degree C for three minutes to cut the DNA which has TaqI site with no mutations. After repeating this cycle five times, the sample was treated with uracil-DNA glycosylase to remove the probe which has no mutation at the TCGA site. Quantitative PCR was carried out at the end to measure the number of the target sequence recovered and the number of the fragment which has mutation at the TCGA site.
Sensitivity of this method was not enough to detect spontaneous mutation frequency on bacterial genomic DNA. One big reason to be considered would be pseudo positive clones after incomplete digestion with Taq1 enzyme. The RMC method will be subjected into mitochondrial DNA. Less
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